The next decade is going to be a telling one for those working in pursuit of cardiac optogenetics. Initial proofs have been demonstrated, the optical tools have been developed and scientific evidence has grown to a point where the tremendous potential of this technique is clear. Cardiac optogenetics holds out the promise of a wide range of clinical possibilities from halting heart attacks and treating arrhythmia to displacing the need for animal testing of drugs. But there are doubters, even among the researchers themselves, that the remaining hurdles may, in the end, prove too complex to bring this promising technique into the clinic. All-optical cardiac electrophysiology allows for parallel testing of many cells at the same time, which is used to establish if a particular drug causes any cardiac side effects. Adapted from A. Klimas et al., Nature Communications, 2016. Courtesy of Aleks Klimas and Emilia Entcheva, GWU. Cardiac optogenetics was first demonstrated in 2010 by Bruegmann et al. in a paper published in the journal Nature Methods. In the research, pulses of light were applied to the hearts of mice in which light-sensitive proteins had been specifically introduced. The process, known as transgenesis, uses gene transfer — in this case to insert proteins that render the heart muscle cells (cardiomyocytes) sensitive to blue light. “Cardiac optogenetics is an exciting new area of research, not only for investigation of mechanisms involved in heart pathologies, but eventually also for treating cardiac arrhythmia in patients,” said Dr. Philipp Sasse of the department of physiology at the University of Bonn, Germany, one of the scientists who wrote the pioneering 2010 paper. “Since our first report in 2010 using light to stimulate cardiomyocytes in vitro and to pace the heart from transgenic mice in vivo,” he said, “the field of cardiac optogenetics has emerged and is still expanding.” Among the most significant developments since 2010 is optogenetic defibrillation in transgenic mice, which can terminate a lethal cardiac arrhythmia using light. Optogenetic stimulation has marked advantages over current electrical approaches such as low energy consumption, cell-specific stimulation and high spatial precision. However, the transgenesis process, which is the essential first step in utilizing optogenetics, creates its own set of problems. The gene transfer of light-sensitive proteins to the heart often triggers an immune response. Although Sasse has shown that the process causes no side effects in mice, it is still unclear whether this will be the case in humans or if efficacy will be maintained in the long term. Despite this, research into the possibilities of cardiac optogenetics continues. At George Washington University, professor Emilia Entcheva, together with professor Gil Bub at McGill University, is exploring optical cardiac electrophysiology in which optics is used to both stimulate cells and record from them. Independent testing of cells The hope is that scaling up the process will provide an all-optical method for wave steering in vitro or in vivo. This would allow fast, simultaneous and independent testing of thousands and millions of cardiac cells — something that has not been an option previously. “Having these all-optical tools also enables us to think of closing the feedback loop for some new ways to control cardiac activity and arrhythmias, in particular,” Entcheva said. Another application of all-optical cardiac electrophysiology, said Entcheva, is for parallel testing of drug action using heart cells in culture. These can be patient-specific cells derived noninvasively from the blood of a patient that has been used to generate stem cells, which in turn have been differentiated into heart cells. “As these fields advance rapidly, we see the stem cell technology and the cardiac optogenetics field displacing the need for animal testing of drugs down the line,” Entcheva said. “Importantly, the merger of these can offer for the first time a viable path to personalized medicine, when it comes to testing for cardiac or neural effects of drugs. Our recently published OptoDyCE platform illustrates how this can be done.” In other work, Entcheva and Bub are exploring the potential of controlling cardiac activity by steering waves of activity in the heart. By using optogenetics and patterned light, they found that they could precisely alter the speed of cardiac waves at desired locations, and change their directionality and the paths that they follow. And, all this can be done with relatively low-intensity light. An all-optical feedback-control system can be used to steer cardiac waves. Shown in color is a cardiac reentrant wave that was manipulated by light to change its direction of rotation. Adapted from R.A.B Burton et al., Nature Photonics, 2015. Courtesy of Gil Bub, McGill and Emilia Entcheva, GWU. “While the path to seeing this optogenetics-based technology in humans is long, we believe in its great value as a fast biological computer — to simulate multiple scenarios in cardiac tissue in the lab and inform decisions about the design of a new-generation cardiac device, be it purely electrical or optical,” Entcheva said. At Stanford University, Dr. Oscar Abilez is working on a project funded by the National Institutes of Health to create an all-optical electrophysiological engineered heart muscle (EHM) system. Engineered from around 1.5 million cardiomyocytes, 0.5 million fibroblasts and collagen, the mix forms tissue-like structures that contract on their own. The goal is to both control the beating of EHMs with light and also to detect the engineered muscle’s electrophysiological response to light. Such “hearts-in-a-dish” could prove critical for disease modeling and drug toxicity testing. Optogenetic engineered heart muscle (EHM) will be used for disease phenotype recapitulation and rescue. First, optogenetic human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) will be engineered to be responsive to light and optically output action potentials (APs) (a). Second, these hiPSC-CMs will be used along with other cardiovascular cell types (e.g., fibroblasts, vascular cells) to create EHM. Finally these EHM will be used to model disease for both diagnostic and therapeutic purposes (b). Red-ChR2: red light-responsive channelrhodopsin-2; hiPSC: human induced pluripotent stem cell; CM: cardomyocyte; hv: light; GEVI: genetically encoded voltage indicator; AP: action potential. Courtesy of Oscar Abilez, Stanford University. “This system will allow the nondestructive, longitudinal control and monitoring of EHMs that cannot be done with current techniques,” Abilez said. “In my opinion, cardiac optogenetics is still in its infancy and the next decade will bring about interesting and useful developments in this field.” Simulated hearts also can be engineered in individual cells, according to Adam Cohen at the Howard Hughes Medical Institute, Harvard University, and co-founder of Q-State Biosciences. In Cohen’s work, stem cell technology, optogenetics and advanced optical imaging are combined to create in vitro models of neuronal and cardiac diseases. “We used voltage and calcium imaging to study the onset of cardiac activity during embryonic development in the zebrafish: The fish egg started as a single cell and 24 hours later it had a beating heart,” Cohen said. “We asked: How does this organ start up?” High time-resolution images show the propagation of voltage (red) and calcium (blue) over the heart during a cardiac cycle. The ventricle (V) and atrium (A) are labeled. Courtesy of Jennifer Hou, Harvard University. The next natural question was: What causes cells to beat irregularly? To begin to answer this, Cohen’s team took some simple cells that were electrically inert and genetically engineered them to produce cardiac-like electrical spikes. Under slow or moderate pacing rates, the cells beat in a regular rhythm; under fast pacing, however, the cells undergo a transition to an arrhythmia-like irregular beating pattern. The research is being used to reveal subtleties about dynamic systems, including what kinds of stimuli can trigger arrhythmias and how one might switch irregularly firing cells back into a regular rhythm. The research also provides a simple system for testing whether candidate drugs affect the ion channels that are important for maintaining regular heart function. A major concern in the pharmaceutical industry is to avoid cardiac side effects: Nobody wants to develop a drug that cures your headache, but stops your heart. These measurements can provide an early warning that a candidate drug might have a cardiac side effect, which is important to know before testing the drug in people. Cardiac optogenetics pioneer Sasse said that the past seven years have seen a slow but steady progress in the field. Whether or not this progress will result in significant clinical translation depends on continued research into this promising technology. “Although all the current reported in vivo experiments in rodents and in silico stimulations suggest successful optogenetic cardiac pacing after cell or gene delivery,” he said, “further in vivo experiments on nontransgenic large animals are clearly needed to answer the question whether an optogenetic cardiac pacemaker or defibrillator is future science or will remain fiction.” Optogenetics: For the head or the heart? The term optogenetics was first coined in 2006 by professor Karl Deisseroth at Stanford University as part of his groundbreaking research in neuroscience. The name captures the concept well, and it stuck — the process involves optics and light, combined with a genetic modification. In the face of skeptics, Deisseroth managed to use light to make adjustments to specific neurons. Optogenetic research has since exploded in the neuroscience community. But the same cannot be said for its translation to the cardiac field. That’s surprising, given the fundamental parallels that exist between neurons and the brain and cardiac cells and heart tissue: Both systems are electrically active and can be stimulated or controlled in similar ways. One of the biggest champions of cardiac optogenetics, Emilia Entcheva at George Washington University, highlights the overcautious nature of cardiac scientists. “The culture of the cardiac research field is much more conservative compared to neuroscience, which makes it slower to adopt new tools,” she said. “It has not seen the explosive growth that neuroscience experienced after the first successful reports. Nevertheless, it is definitely an area of high interest at the moment and multiple research groups look for ways to adopt it.” For many, the time is ripe, as several transgenic mouse models with cardiac relevance have been made available for purchase. Additionally, the neuroscience community and the optics/photonics community have already generated the arsenal of tools to instantly apply to the heart. “The cardiac community still faces more challenges than brain researchers when it comes to routinely applying the techniques in living, freely moving animals,” continued Entcheva. “It is very hard to provide stable and effective light access to the heart, much harder than to do so to the brain.” Just as for the brain, the first step is to genetically modify the tissue to make it responsive to light of a particular wavelength. As with brain cells, gene therapy can be used to make heart cells express light-sensitive proteins that naturally exist in lower organisms, such as algae or bacteria. Developments in neuroscience have helped generate a number of these proteins (called opsins), ensuring that they are nontoxic and suitable for use in mammalian cells, including human. If the goal is to optically pace the heart, opsins are used that reliably trigger activity when optical pulses are delivered. If the goal is to halt abnormal cardiac activity (arrhythmias), stronger light is used in combination with the appropriate activating opsins. Additionally, light-sensitive pumps such as halorhodopsin can be used to completely stop cardiac activity. Each of these opsins imparts sensitivity to a particular wavelength from the visible spectrum; most common are optogenetic tools that respond to blue light (470 nm), but there are proteins that can make cells responsive to red or near-infrared light, which may be desirable for deeper tissue penetration — as needed in the heart. The light levels required to control genetically modified cardiac tissue are quite low. In fact, simple light-emitting diodes can do the job. But to have longer-term control in the intact body, miniaturized implantable devices will be needed that can be easily recharged. Neurons are particularly well-suited to optogenetic research because of fast timescales of signaling, said Stanford neuroscientist Deisseroth. But muscle cells such as those in the heart are over 100 times slower. Nevertheless, he said, when it comes to the heart, the optogenetic method is far more precise than pharmacological or electrical methods and further research could offer some real benefits. “I believe that any optogenetic advance in basic science, for [the] heart or brain, will advance the application of any kind of treatment — [be it] drug, electrical, or [a] combination,” Deisseroth said.